Methods for preventing plastic-induced degradation of biologicals

ABSTRACT

The present invention provides methods for protecting a virus contained in a bioprocess bag from surface-induced degradation, thereby improving the compatibility of viruses with these plastics by preserving quantity and potency of the contained viruses during storage in bioprocess bags over a period of time. It was shown herein that viruses can quickly degrade when stored in bioprocess bags and that the addition of a β-cyclodextrin to a virus-containing solution stored in a bioprocess bag unexpectedly prevents the plastic surface-induced degradation of said virus.

FIELD OF THE INVENTION

The present invention relates to methods for preventing surface-induceddegradation of biologicals in bioprocess bags. In particular, it relatesto the use of β-cyclodextrins for preventing deterioration of viruses insolutions contained in bioprocess bags. The methods of the presentinvention improve the compatibility of said viruses with plasticbioprocess bags by preserving quantity, structure, potency and qualityof the contained viruses.

BACKGROUND OF THE INVENTION

With a clear trend in pharmaceutical industry towards disposablesystems, an ongoing challenge in the field of viruses is to generatemethods for manufacturing and bulk storage of liquid compositions thatcontain said viruses. Especially wherein said viruses are stabilized forlonger periods of time within a realistic storage temperature range forpharmaceutical products, such as from about 2° C. to about 8° C. Forpractical and logistical reasons, bioprocess bags, which are plasticcontainers, are often used to store viruses. Said large structures oftendeteriorate inside these plastic containers, as a consequence ofmechanisms that are largely unknown.

Biological activity of a virus depends upon the conformational integrityof at least a core sequence of amino acids. Unlike traditional organicand inorganic small molecules, viruses are highly complex biologicalstructures and minor chemical or physical stressors can contribute tothe degradation of the virus. Compatible primary packaging for bulkstorage is of crucial importance to ensure a reasonable shelf-life, butgiven the nature of the disposable storage containers used in theindustry, this poses particular challenges. Viruses contained in abioprocess bag tend to lose potency as a result of surface-induceddegradation.

Accordingly, there is a need in the art to find methods for improvingthe compatibility of viruses with bioprocess bags that are used formanufacturing and storage of viruses. In particular there is a need formethods that protect viruses contained in bioprocess bags, fromsurface-induced degradation and therewith improve shelf life of saidviruses during processing and storage in bulk.

SUMMARY OF THE INVENTION

We have found and describe herein, methods for protecting a viruscontained in a bioprocess bag from surface-induced degradation. Thesemethods improve the compatibility of viruses with plastic bioprocessbags by preserving quantity, structure, potency and quality of the virusas compared to previously disclosed methods. Remarkably, the addition ofa β-cyclodextrin to any solution comprising viruses, contained in abioprocess bag, resulted in an outstanding preservation of structure andpotency of said viruses, therewith unexpectedly improving the overallcompatibility of said viruses with the plastic surface of the bulkstorage container as compared to the same solution without theβ-cyclodextrin.

The present invention therefore relates to methods for protecting avirus from surface-induced degradation wherein said virus is containedin a solution in a bag comprised of a plastic, and wherein said methodcomprises the step of adding a β-cyclodextrin to said solution at aconcentration between about 1% (w/w) to about 30% (w/w). In a preferredembodiment of the present invention, said cyclodextrin is aβ-cyclodextrin selected from the group of dimethyl-β-cyclodextrin,2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin,3-hydroxypropyl-β-cyclodextrin and trimethyl-β-cyclodextrin. In an evenmore preferred embodiment, said β-cyclodextrin is2-hydroxypropyl-β-cyclodextrin.

In a more preferred embodiment according to the present invention, thefluid contact layer of the plastic is selected from the group ofEthylene Vinyl Acetate, Polyethylene, Polyamide and Polyethylene. Morepreferably said layer is Ethylene Vinyl Acetate. In certain embodimentssaid plastic includes a gas barrier. Preferably said gas barrier is madefrom Ethyl Vinyl Alcohol. In a preferred embodiment according to thepresent invention said bag is a bioprocess bag. In another preferredembodiment according to the present invention, said virus is anadenovirus.

In a more preferred embodiment, the present invention relates to amethod for protecting an adenovirus from surface-induced degradation,wherein said adenovirus is contained in a solution in a bag comprised ofplastic, and wherein said method comprises the step of adding aβ-cyclodextrin to said solution at a concentration between about 1%(w/w) to about 30% (w/w). Preferably said bag has a fluid contact layercomprised of Ethylene Vinyl Acetate bag. More preferably saidβ-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

In a more preferred embodiment, the present invention relates to amethod for protecting an adenovirus from surface-induced degradation,wherein said adenovirus is contained in a solution in a bag comprised ofEthylene Vinyl Acetate, wherein said method comprises the step of adding2-hydroxypropyl-β-cyclodextrin to said solution at a concentrationbetween about 1% (w/w) to about 30% (w/w).

It was demonstrated herein, for the first time, that the addition of aβ-cyclodextrin can provide protection against surface-induceddegradation of a virus when stored in a bioprocess bag. The presentinvention therefore also relates to the use of a β-cyclodextrin forprotecting a virus from surface-induced degradation wherein said virusis contained in a solution which is contained in a plastic bioprocessbag.

Preferably said β-cyclodextrin is selected from group ofdimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin andtrimethyl-β-cyclodextrin. In an even more preferred embodiment, saidβ-cyclodextrin is a 2-hydroxypropyl-β-cyclodextrin.

In a more preferred embodiment according to the present invention, theplastic is a polymer. Preferably said plastic is selected from the groupof Ethylene Vinyl Acetate, Polyethylene, Polyamide and Polyethylene. Incertain embodiments said plastic includes a gas barrier. Preferably saidgas barrier is Ethyl Vinyl Alcohol.

In another more preferred embodiment according to the present invention,said virus is an adenovirus.

The present invention also relates to the use of a2-hydroxypropyl-β-cyclodextrin for protecting an adenovirus fromsurface-induced degradation wherein said adenovirus is contained in asolution which is contained in a bioprocess bag.

The present invention also relates to the use of a2-hydroxypropyl-β-cyclodextrin for protecting an adenovirus fromsurface-induced degradation wherein said adenovirus is contained in asolution which is contained in an Ethylene Vinyl Acetate bioprocess bag.

DESCRIPTION OF THE FIGURES

FIG. 1: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by intrinsic protein fluorescence. Mean valuesand standard deviations are shown, with lines indicating the trends.

FIG. 2: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by hexon content by RP-UPLC. Mean values andstandard deviations are shown, with lines indicating the trends.

FIG. 3: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by protein concentration by absorbance at 280nm. Mean values and standard deviations are shown, with lines indicatingthe trends.

FIG. 4: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by adenovirus titer by vp-QPCR. Mean values andstandard deviations are shown, with lines indicating the trends.

FIG. 5: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by adenovirus concentration by AEX-UPLC. Meanvalues and standard deviations are shown, with lines indicating thetrends.

FIG. 6: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles and solid lines) or glass vials (open diamonds anddotted lines), when stored at 5° C. (left) or 25° C. (right) for up tofour weeks, as monitored by potency by QPA. Mean values and standarddeviations are shown, with lines indicating the trends.

FIG. 7: Compatibility of Ad26 in formulation 1 with plastic Flexboy bags(closed circles) or glass vials (open diamonds) during one freeze/thawcycle (room temperature to −70° C. and back, at 0.032° C./min), asmonitored by intrinsic protein fluorescence (A) and hexon content byRP-UPLC (B). Mean values and standard deviations are shown.

FIG. 8: Compatibility of Ad26 in formulation 1 with (from left to right)Flexboy® bioprocessing bags (Sartorius Stedim Biotech GmbH), HyQ Cx5-14bioprocessing bags (Thermo Fisher), PureFlex Mobius bioprocessing bags(Millipore), or glass vials (Nuova Ompi). Formulations were stored at 5°C. (closed squares) or 25° C. (open triangles) for up to 24 weeks, andcompatibility was monitored by potency by QPA. Mean values and standarddeviations are shown, with linear fits to the data, with 95% confidenceintervals shaded grey.

FIG. 9: Compatibility of Ad26 in formulations 1, 2 and 3 (from left toright), either without HBCD (open circles and dashed lines) orsupplemented with 5% (w/w) HBCD (crosses and solid lines), with plasticFlexboy bags during storage at 25° C. for up to one week, as monitoredby intrinsic protein fluorescence. Mean values and standard deviationsare shown, with lines indicating the trends.

FIG. 10: Compatibility of Ad26 in formulations 1, 2 and 3 (from left toright), either without HBCD (open circles and dashed lines) orsupplemented with 5% (w/w) HBCD (crosses and solid lines), withbioprocess bags during storage at 25° C. for up to one week, asmonitored by hexon content by RP-UPLC. Mean values and standarddeviations are shown, with lines indicating the trends.

FIG. 11: Compatibility of Ad26 in formulations 1 and 2 (from left toright), either without HBCD (open circles and dashed line) orsupplemented with 5% (w/w) HBCD (crosses and solid line), withbioprocess bags during storage at 25° C. for up to one week, asmonitored by Adenovirus particle concentration by CE. Lines show linearfits to the data, with 95% confidence intervals shaded grey. Formulation3 without HBCD was degraded to such an extent after contact with thebioprocess bag that no virus particle concentration could be determined.

FIG. 12: Compatibility of Ad26 in formulation 2 supplemented with 0,0.2, 1 or 5% (w/w) HBCD with plastic Flexboy bags after 8 days storageat 25° C., as monitored by intrinsic protein fluorescence. Individualdata points are shown, with a line indicating the trend.

FIG. 13: Compatibility of Ad26 in formulation 2 supplemented with 0,0.2, 1 or 5% (w/w) HBCD with plastic Flexboy bags after 8 days storageat 25° C., as monitored by protein concentration by absorbance at 280nm. Individual data points are shown, with a line indicating the trend.

FIG. 14: Compatibility of Ad26 in formulation 2 supplemented with 0,0.2, 1 or 5% (w/w) HBCD with plastic Flexboy bags after 8 days storageat 25° C., as monitored by hexon content by RP-UPLC. Individual datapoints are shown, with a line indicating the trend.

FIG. 15: Compatibility of Ad26 in formulation 2 supplemented with 0,0.2, 1 or 5% (w/w) HBCD with plastic Flexboy bags after 8 days storageat 25° C., as monitored by Adenovirus particle concentration by CE. Theline shows a linear fit to the data, with the 95% confidence intervalshaded grey.

FIG. 16: Compatibility of Ad26 in formulation 2 supplemented with 0,0.2, 1 or 5% (w/w) HBCD with plastic Flexboy bags after 8 days storageat 25° C., as monitored by potency by QPA. The line shows a linear fitto the data, with the 95% confidence interval shaded grey.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned previously, there is a need to find methods for protectinga virus contained in a bioprocess bag from surface-induced degradation,thereby improving the compatibility of viruses with process and bulkstorage materials by preserving quantity and potency of the containedviruses. It was shown herein that the viruses quickly degrade inbioprocess bags and that the addition of a β-cyclodextrin like2-hydroxypropyl-β-cyclodextrin (all molar substitutions, HBCD) to avirus in solution stored in a bioprocess bag surprisingly prevents theplastic surface-induced degradation of said virus.

We have found and describe herein, methods for preserving viruses inbioprocess bags. In particular, these methods protect viruses fromsurface-induced degradation when said viruses are contained in asolution in a bag comprised of a plastic. These methods improve thebiological compatibility with plastics by preserving quantity andpotency and quality of the virus as compared to previously disclosedmethods regardless of the formulation matrix used. Examples of theviruses for which the methods of the present invention are suited are,but not limited to, (active) viruses, vaccines, non-enveloped viruses,enveloped viruses, virus-like particles, recombinant viruses, andinactivated and attenuated viruses.

In a preferred embodiment of the present invention, the virus is arecombinant adenovirus. The construction and propagation of adenoviralvectors is well understood in the art and involves the use of standardmolecular biological techniques, such as those described in, forexample, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2ded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watsonet al., Recombinant DNA, 2d ed., Scientific American Books (1992), U.S.Pat. No. 6,492,169 or in WO 03/104467 U.S. Pat. Nos. 5,559,099,5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541,5,981,225, 6,040,174, WO 96/26281, WO 00/03029, and Thomas Shenk,“Adenoviridae and their Replication”, which is incorporated here byreference. In certain embodiments of the present invention, serotypes ofhuman adenovirus include any one of serotypes 2, 4, 5, 7, 11, 26, 34,35, 36, 48, 49 or 50 or any hybrid or mutated adenovirus serotypes. In apreferred embodiment of the present invention the recombinant adenovirusis from human adenovirus serotype 5, 26 or 35. In further embodiments,the adenovirus of the invention is a simian adenovirus, preferably achimpanzee or gorilla adenovirus. These adenoviruses generally have alow seroprevalence and/or low pre-existing neutralizing antibody titersin the human population. In further embodiments, the adenovirus of theinvention further comprises heterologous nucleic acid. Suitableheterologous nucleic acid is well known to the skilled person, and forinstance may include transgene open reading frames, for instance openreading frames coding for polypeptides against which an immune responseis desired when the vector is used for vaccination purposes, e.g.transgenes suitable to generate an immune response against malaria (seee.g. WO 2004/055187), HIV, Ebola, RSV, HPV, Zikavirus, HSV, Tuberculosis(see e.g. WO 2006/053871), certain viruses, etc, all well known to theskilled person. In fact, the nature of the transgene is not critical tothe current invention, it may be any heterologous nucleic acid sequence,and hence needs no further elaboration here. A few references disclosethe use of cyclodextrins as excipient for the formulation ofadenoviruses. WO029024 discloses hydroxypropyl-β-cyclodextrin as part ofa large list of possible lyoprotectants used for preparing a freezedried formulation. WO029024 relates to a freeze dried composition asopposed to a liquid composition as disclosed in the present invention.The advantage of a liquid formulation is that it is less expensive, andthe handling before administration is less time consuming and less proneto clinical dosing or reconstitution mistakes. Furthermore, scale up oflyophilization processes can be a cumbersome endeavor. Renteria et al.identifies HBCD as one of the additives used to promote the stability ofcertain proteins and to avoid aggregation during nasal administration.Renteria et al. discloses the use of hydroxypropyl-β-cyclodextrin in thecontext of a formulation appropriate for nasal administration enhancingmucosal uptake. WO2015/04002 discloses complex multi-componentvirus-containing compositions containing HBCD as one of the components.The virus-containing compositions which comprise HBCD and which areexemplified in WO2015/04002, are all contained in glass vials.WO2015/04002 is silent about the incompatibility of Adenoviruses withplastic bioprocess bags.

HBCD is typically used to increase the solubility of small molecules(e.g. diclofenac or ibuprofen). This has to do with its unusualstructure. The HBCD molecule is a torus shaped ring with a polarhydrophilic outside and an apolar hydrophobic cavity due to the spatialdistribution of its external hydrophilic properties. The secondary OHgroups are on the opposite edge. These hydroxyl groups give HBCD itsexternal hydrophilic properties. The inside of the HBCD ring is small indiameter (much smaller than a viral particle) and fits only smallmolecules. It is composed of a surface of hydrophobic hydrogens as wellas glycosidic ether-like oxygen. As a consequence of this particularstructure, HBCD is used to encapsulate or entrap very small moleculessolubilizing them to form the so-called inclusion compounds. Thesestructural features of HBCD do not render HBCD an obvious choice for usein solving incompatibility problems in bioprocess bags.

The term “compatibility” refers to the ability of two materials orcomponents to exist in contact with or next to each other, withoutnegatively influencing attributes such as quantity, structure, potencyor quality of one or both of the components. As used herein it refers tothe relative resistance to degradation of virus particles in contactwith or next to the surface of a bioprocess bag or other packagingmaterial.

The term “potency” as used herein refers to a measure of adenovirusactivity expressed in terms of infectious units measured in a cell-basedpotency assay, which is described hereunder.

The term “by-product” includes undesired products, which detract ordiminish the proportion of therapeutic/prophylactic adenovirus in agiven formulation. Typical by-products include aggregates of theadenovirus and fragments of the adenovirus, resulting from e.g. proteindenaturation, deamidation, hydrolysis or combinations thereof.Typically, aggregates are complexes that have a molecular weight greaterthan the isolated virus particle.

A method that protects a virus from surface-induced degradation as usedherein and which thereby improves the compatibility with plastics, is amethod that preserves the physical and/or chemical integrity and/orbiological activity of the virus upon storage in a bioprocess bag. Thiscan be assessed by determining different characteristics such as thepotency, and/or other quality aspects of the virus in the bioprocessbags over a period of time and under certain storage conditions. Thesecharacteristics of said virus can be measured at elevated temperatures(predictive for real-time temperatures) or under other stressconditions, for instance viruses can be subjected to incubation at 25°C. in order to study the effects of different conditions maximizingbiological shelf-life. Said characteristics which determine thedegradation of viruses may be determined by at least one of the methodsselected from the group consisting of visual inspection, virus particlequantitative polymerase chain reaction (vp-QPCR), QPCR-based PotencyAssay (QPA), Reverse Phase High Performance Liquid Chromotography(RP-UPLC), AEX-UPLC, Intrinsic Fluorescence and protein concentration byabsorbance at 280 nm.

Virus Particle Quantitative Polymerase Chain Reaction (vp-QPCR)

The vp-QPCR was developed for the quantification of adenovirus particlesusing primers that target a 100 bp region of the CMV promoter of thetransgene cassette present within the adenovirus vector. Briefly, thisQPCR method relies on the exonuclease activity of Taq polymerase, whichresults in degradation of a specific fluorescent probe annealed in themiddle of the 100 bp amplicon. The probe is covalently linked to a lightemitter and a quencher, and its degradation frees the emitter from thequencher with a consequent fluorescence emission proportional to theamount of template. Quantitative values are obtained from the thresholdcycle (Ct), the cycle at which an increase in fluorescence signalexceeds a threshold value. The threshold for detection of DNA-basedfluorescence is set slightly above background. The number of cycles atwhich the fluorescence exceeds the threshold is called the thresholdcycle (Ct) or, according to the MIQE guidelines, quantification cycle(Cq) (Bustin et al, 2009). During the exponential amplification phase,the target DNA sequence doubles every cycle. For example, a DNA sampleof which the Ct precedes that of another sample by 3 cycles contained2³=8 times more template. Consequently, a higher Ct value represents alower amount of target DNA and a lower Ct value represents a highavailability of target DNA. Absolute quantification can be performed bycomparing a standard curve generated by a serial dilution of a stockadenovirus of which the concentration has been determined by the opticaldensity at 260 nm (OD₂₆₀). The Ct value of the test material is plottedagainst the Ct values of the standard curve, which generates an accurateand precise number of vector particles.

When used as readout after incubation on E1 competent cells (QPA, seebelow), more degraded samples will lead to higher delta (t=0 subtracted)Ct values and more stabilizing formulations will lead to lower Ctvalues.

QPCR-Based Potency Assay (QPA)

To quantify adenovirus potency, the QPA combines QPCR with a tissueculture-based infectivity assay. The assay is based on the experimentalobservation that the appearance of newly synthesized viral DNA is veryrapid after inoculation of a cell-monolayer, and is proportional to thevirus input concentration over a large range of multiplicity ofinfection (MOI). Dilutions of samples (non-endpoint diluted) areinoculated onto HEK293 cell monolayers in a 96-well plate. The infectionis allowed to proceed for 3 hours at 35° C. Wells are aspirated andreplenished with medium that does not contain adenoviruses. Plates areincubated for an additional 42 hours prior to cell lysis by means ofTriton X-100 solution and a single freeze/thaw step in order to releaseadenovirus DNA. A QPCR is performed on diluted cell lysates according tothe method described above. The infectivity titer is calculated bycomparison to a standard curve generated by the Ct values of a sample ofknown infectivity, which is determined by endpoint titration.Alternatively, the delta potency can be expressed directly as Ct valuessince the infectivity titer, or potency, is directly correlated to theCt values. Especially in comparing relative differences in potencybetween formulations, this is a quick and reliable method.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography (RP-UPLC)

In order to determine several quality attributes of an adenovirus, suchas presence and (relative) quantity of adenoviral and other proteins,one can analyze adenoviral protein profiles by Reversed-PhaseUltrahigh-Performance Liquid Chromatography (RP-UPLC). UPLC separatescomponents of a mixture by using a variety of chemical interactionsbetween the sample, the mobile phase (a buffer or solvent) and thestationary phase (a chromatographic packing material in a column). Ahigh-pressure pump moves the mobile phase through the column, while theanalyte partitions between the two phases, so that its elution dependson its relative affinities for the stationary and mobile phases. Adetector measures the retention times (t_(R); time between sampleinjection and the appearance of the peak maximum) of the molecules usinge.g. UV absorbance detection.

The separation mechanism of RP-UPLC is based on differences inhydrophobicity. In the adenovirus protein profiling method, thenon-polar stationary phase is made up of hydrophobic alkyl C4 chains,while the mobile phase consists of a water/acetonitrile/TFA mixture withincreasing hydrophobicity. Adenoviral particles dissociate into theirconstituent proteins, which initially interact with the stationaryphase, and subsequently elute at a retention time depending on theirhydrophobic surface area. The retention time and amount of each proteinare monitored by UV absorbance detection at 280 nm. A typical adenoviralRP-UPLC profile consists of 10 or 14 proteins, including core protein(VII), penton base (III) and hexon (II).

Anion-Exchange Ultrahigh-Performance Liquid Chromatography (AEX-UPLC)

In order to quantify the number of adenovirus particles, one canseparate the virus particles from the matrix by Anion-ExchangeUltrahigh-Performance Liquid Chromatography (AEX-UPLC), and quantifythem by UV absorbance.

UPLC separates components of a mixture by using a variety of chemicalinteractions between the sample, the mobile phase (a buffer or solvent)and the stationary phase (a chromatographic packing material in acolumn). A high-pressure pump moves the mobile phase through the column,while the analyte partitions between the two phases, so that its elutiondepends on its relative affinities for the stationary and mobile phases.A detector measures the retention times (t_(R); time between sampleinjection and the appearance of the peak maximum) of the analytes usinge.g. UV absorbance detection.

In the AEX-UPLC method for adenovirus particle quantification, thenegatively charged particles are captured via their interaction with apositively charged column containing quaternary ammonium cations.Increasing salt concentration in the mobile phase weakens theelectrostatic interactions, so that the viral particles elute at aretention time specific for their serotype, separate from matrixcomponents and impurities. The particles are subsequently quantified byUV absorbance at 214 nm.

Capillary Zone Electrophoresis (CE)

Capillary zone electrophoresis (CE) is used to determine the Adenovirusparticle concentration. In this method, a sample containing virusparticles moves through a small diameter capillary via electroosmoticflow. The various components in the sample have differentelectrophoretic mobilities, because of size, charge and frictionaldifferences. This causes the different components to separate intobands.

Upon elution, the Adenovirus particle peak is detected and quantified byabsorbance at 214 nm. The signal is converted into a concentration via acalibration standard.

Intrinsic Fluorescence Assay

The adenoviral capsid proteins contain aromatic amino acids that reemitlight after excitation, in particular tryptophan and to a lesser extenttyrosine and phenylalanine. The emission maximum and quantum yield oftryptophan depend strongly on the polarity of its environment. In apolar, aqueous environment (e.g. the surface of a globular protein) thequantum yield is relatively low, while in an apolar environment (e.g.the inside of an aggregate) the quantum yield increases. This featuremakes tryptophan fluorescence a useful tool for studying proteinconformational change, aggregation, and molecular interactions.

In the intrinsic fluorescence assay, samples are transferred intriplicate to a UV-transparent, flat-bottom microplate. The plate iscovered with a UV-transparent seal. Tryptophan fluorescence is measuredby a microplate reader using an excitation filter with a centerwavelength of 280 nm and a bandwidth of 10 nm, and an emission filterwith a center wavelength of 340 nm and a bandwidth of 10 nm. Bottomoptic is used to minimize the influence of the seal and the meniscusshape.

The fluorescence intensity is known in the art to be a sensitive measureof adenovirus degradation. Either an increase or a decrease may beobserved upon stress, depending on the nature of the changes occurringin the sample. Protein unfolding and capsid dissociation is expected tolead to a decrease in intrinsic fluorescence, and aggregation isexpected to lead to an increase. The precision of the assay is <5% (CV%) in the range used.

The obtained fluorescence for stressed samples should always be comparedto the control samples. Since an increase or decrease after appliedstress is dependent on the degradation pathway and specific for eachActive Pharmaceutical Ingredient (API), it cannot be predicted. A change(higher or lower) compared to the t=0 samples is indicative of a lessstable formulation. Stressed samples remaining close to the t=0 samplevalues are more stable.

Protein Concentration by Absorbance at 280 nm

The adenoviral capsid proteins contain aromatic amino acids that absorbultraviolet light around 280 nm, in particular tryptophan and to alesser extent tyrosine and phenylalanine. The absorbance is linearlycorrelated with the number of amino acids in the optical pathlength, andthereby to the protein concentration, according to the Beer-Lambert law.Since the viral particles also scatter light, resulting in awavelength-dependent apparent absorbance, the signal is first correctedfor this scattering signal to obtain a true absorbance signal. Theabsorbance at 280 nm is subsequently either translated to a proteinconcentration via a calibration curve, or directly compared betweensamples as a quantitative measure of protein concentration.

A virus is compatible with a primary bulk container such as a bioprocessbag, if amongst others, it shows minimal loss (i.e. 0.3 log/2 years) interms of quantity and potency, and displays no major modifications.Additionally, no signs of aggregation, precipitation, change of colorand/or clarity upon visual examination should be observed.

“About” as used in the present application means ±10%, unless statedotherwise.

“Plastic” as used in the present application means: synthetic orsemi-synthetic malleable (co-)polymer of one or more organic molecules.This includes films consisting of multiple layers, such as a fluidcontact layer, a gas barrier layer, and outer layers, of which at leastthe fluid contact layer is a plastic. Examples of plastics used in thepresent application are Ethylene Vinyl Acetate (including monolayer),(Ultra-Low Density) Polyethylene, Polyamide and Polyethylene.

“Bag” as used in the present application means: a bioprocess containermade of plastic, which may be used during the manufacturing process ofviruses, in order to hold process intermediates or (bulk) viruses, or asprimary packaging. Examples of bags as used in the present applicationare e.g. Flexboy bags, HyQ bags, PureFlex bags, wave bags, bioreactors,etc. These bags are commonly referred as bioprocess bags. The term “bag”and “bioprocess bag” can interchangeably be used within the presentapplication.

“polymer” as used in the present application means: a large molecule, ormacromolecule, composed of many repeated subunits (monomers).

“co-polymer” as used in the present application means: two or moredifferent monomers united together.

In a preferred embodiment of the invention, the bioprocess bag is madeof a polymer. In a more preferred embodiment of the invention, saidpolymer is selected from the group of Ethylene Vinyl Acetate (includingmonolayer), (Ultra-Low Density) Polyethylene, Polyamide andPolyethylene.

“Cyclodextrin” as used in the present application means: group of cyclicoligosaccharides. Among the most commonly used forms are α-, β-, andγ-cyclodextrin, which have respectively 6, 7, and 8 glucose molecules.In accordance with the present invention, the cyclodextrins that offerprotection to viruses against surface-induced degradation in bags areβ-cyclodextrins. Substituted derivatives are also available, likedimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin andtrimethyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. For thelatter group of molecules, “The molar substitution (MS)” as used in thepresent application means: the average number of hydroxypropyl groupsper anhydroglucose unit.

“The degree of substitution (DS)” as used in the present applicationmeans: the number of hydroxypropyl groups per molecule of cyclodextrin.

“gas barrier” as used in the present application means: a layerimpermeable to gas in between two layers of monomers, such as ethylvinyl alcohol.

“w/w” as used in the present application means: “weight for weight” or“weight by weight”, the proportion of a particular substance within amixture, as measured by weight or mass.

The present invention relates to methods for protecting viruses fromsurface-induced degradation in bioprocess bags. These methods improvethe compatibility with plastics by preserving quantity and potency(infectivity) and quality of the viruses as compared to previouslydisclosed methods. Remarkably, the addition of a β-cyclodextrin to asolution comprising a virus, contained in a bioprocess bag resulted inan outstanding preservation of quantity, potency (infectivity) andquality of said virus, therewith improving the overall compatibility ofsaid virus with bioprocess bags as compared to the solution without theβ-cyclodextrin.

The present invention relates to methods for preserving viruses andrelated pharmaceutical products in a solution, preferably for use ingene therapy and/or vaccine applications. The solutions that contain thevirus are appropriate for manufacturing and storage in the 2-8° C. rangewhile also being compatible with parenteral administration. Thesesolutions could also be stored at lower temperatures, e.g. −20° C. orlower, −40° C. or lower, −65° C. or lower, −80° C. or lower. They mayalso be stored at temperatures above 8° C., e.g. 25° C. or even higher.

The solutions used in the present invention provide compatibility toplastic, to the viruses at varying concentrations, and may beadministered to a variety of vertebrate organisms, preferably mammalsand especially humans. The stabilized solutions used in the methods ofthe present invention are e.g. viral-based compositions, which can, forinstance, be administered as a vaccine that may offer a prophylacticadvantage to previously uninfected individuals and/or provide atherapeutic effect.

In a preferred embodiment of the invention, the β-cyclodextrin used inthe methods of the present invention is selected from the group ofdimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin andtrimethyl-β-cyclodextrin. In an even more preferred embodiment, saidβ-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

In a preferred embodiment, the concentration of the β-cyclodextrin inthe virus-containing solution is ranging between about 1% (v/v) and 30%(v/v), e.g. between about 1.5% (v/v) and 25% (v/v), e.g. between about2% (v/v) and 20%, e.g. between about 2.5% (v/v) and 20% (v/v), e.g.between about 3% (v/v) and 15% (v/v), e.g. between about 3.5% (v/v) and10% (v/v), e.g. about 5% (v/v).

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLES Example 1 Experimental Design and Methodology

The compatibility with glass and plastic of an Adenoviral preparationcomprising an Ad26 adenovirus (as described in [1]) was tested. Ad26adenoviruses were filled in Flexboy® bioprocessing bags (SartoriusStedim Biotech GmbH) and in glass vials (Nuova Ompi), at a concentrationof 2.4×10¹¹ VP/mL in the standard formulation 1 (see Table 1). The 50 mLFlexboy® bioprocessing bags were filled with 20 mL of the formulation,while the 3 mL vials were filled with 1.5 mL (in both cases in duplicateper condition and time point). Control samples were taken from theformulation at T=0 before filling the bags and vials.

To evaluate the compatibility of the Adenovirus formulation with plasticand glass surfaces, the bags and vials were incubated at 5±3° C. or at25±2° C. for up to four weeks, with time points at 1, 2, 4, 7 and 28days. At each time point, samples were removed from the bags and vialsand stored at ≤−65° C. until sample analysis.

The compatibility of the Adenovirus formulation with plastic as comparedto glass and as compared to the T=0 control samples, was analyzed by thefollowing methods, as described above: intrinsic protein fluorescenceintensity; hexon protein content by RP-UPLC; total protein content byabsorbance at 280 nm; Adenovirus particle concentration by vp-QPCR;Adenovirus particle concentration by AEX-UPLC; and potency (infectivity)by QPCR-based Potency Assay (QPA). The analyses were performed on allbags and vials at all time points, except for the vp-QPCR analysis,which was performed for a selection of samples only.

In order to further evaluate the unexpected finding that the standardformulation 1 was incompatible with the bioprocess bag (see resultssection), additional contact materials and time points were studied.Ad26 adenoviruses in formulation 1 at a concentration of 3.0×10¹¹ VP/mLwas incubated at 5±3° C. or at 25±2° C. for up to six months, inFlexboy® bioprocessing bags (Sartorius Stedim Biotech GmbH), in HyQCx5-14 bioprocessing bags (Thermo Fisher), or in PureFlex Mobiusbioprocessing bags (Millipore); in all cases, 25 mL of the formulationwas filled into a 50 ml bag. Glass vials (Nuova Ompi) were included as acontrol.

At time points 0, 1, 2, 5, 6, 7 and 14 days, and 1, 2, 3, 4, 5 and 6months, samples were removed from the bags and vials and analyzed forcompatibility using the methods as described above.

Results and Conclusions

FIGS. 1-3 show the compatibility of Ad26 adenoviruses with either glassor plastic in formulation 1. Intrinsic fluorescence shows the proteincontent and conformation, hexon peak area the amount of hexon protein,and absorbance at 280 nm the total protein concentration. The resultsshow that, surprisingly, the amount of (total and viral) proteindecreases when formulation 1 is in contact with a plastic surface,already after 1 day at 5 or 25° C. The decrease is strongest during thefirst week, during which a ca. 20-30% drop in protein content occurs.The degradation is only slightly mitigated at the lower temperature. Theeffect is clearly induced by the presence of the plastic surface, sincethe material does not show these changes in protein content whenincubated in glass vials. This incompatibility of the viruses withplastic is a very unexpected finding, since plastic bioprocessing bagsare commonly used for manufacturing and storage of proteins, viruses andother biologicals.

The virus particle concentration follows a similar trend (FIGS. 4 and5), with a decrease of almost 50% for the samples in contact withbioprocess bags, as compared to no significant decreases for the samplesin glass vials. FIG. 6 shows that infectious particles are affected bythe effect, so that the potency decreases concurrently with the viralproteins and particles in the samples in bioprocess bags.

A similar effect was observed when the samples were subjected tofreeze/thaw stress (FIG. 7). After a single freeze/thaw cycle, thematerial in bags showed a clear decrease in protein content, while thesample in glass indicated that the material itself is resistant to thisstress condition. Frozen storage of the virus material is thus not asolution that can prevent the surface-induced degradation.

To investigate whether the incompatibility is specific for the type ofbioprocess bag used in FIGS. 1-7, several other types of plastic bagswere tested for compatibility as well. FIG. 8 shows the potency,expressed as concentration of infectious particles, of a viruspreparation stored in three types of bioprocess bags, as compared toglass vials. The potency decreases strongly, at both 5 and 25° C., whenthe virus material is in contact with any of the plastics. The virusmaterial stored in glass vials, on the other hand, shows goodcompatibility, and degrades at a much slower rate than in the bioprocessbags at both temperatures. This shows that the incompatibility of theviruses with plastics is independent of the type of plastic, and thatthe problem is not easily mitigated by changing to a different type ofbag.

The results demonstrate that the standard formulation 1 degrades rapidlyupon contact with a plastic surface, and that this incompatibilitycannot easily be mitigated by e.g. a lower temperature, frozen storageor a different type of plastic. This poses a real problem formanufacturing, shipping and storing of such materials, since plasticsare commonly used in single-use bioprocessing bags and storagecontainers.

Example 2 Experimental Design and Methodology

An Adenoviral preparation comprising Ad26 adenoviruses (as described in[1]) was concentrated and reformulated by ultrafiltration/diafiltration(using a Sartoslice set-up with Pellicon XL Biomax 300 filter,Millipore) to formulations 1, 2 and 3 as defined in Table 1. Formulation1 is the standard formulation used in example 1, Formulation 2 is thecommonly used adenovirus formulation as described in literature [2], andformulation 3 is the commonly used buffer PBS. The three formulationsdiffer in pH, buffering species, and presence and concentration of otherexcipients.

Subsequently, half of the material was diluted using the appropriateformulation buffer to 1.0×10¹¹ VP/mL (formulation 1) or 1.5×10¹¹ VP/mL(formulations 2 and 3). The other half was spiked with a stock ofhydroxypropyl β-cyclodextrin (HBCD) in the appropriate formulationbuffer up to a final concentration of 5% (w/w) HBCD, in order to obtainformulations 1, 2 and 3+HBCD (Table 1). These formulations were alsodiluted to the same titers using the appropriate formulation buffer.Control samples were taken from all formulations at T=0 before testingthe compatibility of the material with bioprocess bags.

TABLE 1 Formulations tested in this study. Formulation FormulationFormulation Formulation Formulation 2 (Evans et 2 (Evans et al.Formulation Formulation 3 1 1 + HBCD al. [2]) [2]) + HBCD 3 (PBS)(PBS) + HBCD pH 6.5 6.5 7.4 7.4 7.4 7.4 buffer 20 mM 20 mM 10 mM 10 mM10 mM 10 mM Histidine Histidine Tris and Tris and Na₂HPO₄, Na₂HPO₄, 10mM 10 mM 2 mM 2 mM Histidine Histidine KH₂PO₄ KH₂PO₄ cryoprotectant 5%(w/w) 5% (w/w) 5% (w/w) 5% (w/w) N/A 5% (w/w) Sucrose HBCD and SucroseHBCD and HBCD 5% (w/w) 5% (w/w) Sucrose Sucrose tonicity 75 mM 75 mM 75mM 75 mM 137 mM 137 mM modifier NaCl NaCl NaCl NaCl NaCl, NaCl, 2.7 mM2.7 mM KCl KCl EtOH (% w/w) 0.4 0.4 0.4 0.4 N/A N/A PS-80 (% w/w)  0.02 0.02  0.02  0.02 N/A N/A EDTA (mM) 0.1 0.1 0.1 0.1 N/A N/A other N/AN/A 1 mM 1 mM N/A N/A MgCl2 MgCl2

To evaluate the plastic compatibility of the Adenovirus formulations 1,2 and 3 with or without HBCD, 25 mL of each formulation was filled in 50mL Flexboy® bioprocessing bags (Sartorius Stedim Biotech GmbH), in n=3per formulation. The bags were incubated at 25±2° C., with time pointsat 2 or 3 and at 7 or 8 days. At each time point, samples were removedfrom the bags and stored at ≤−65° C. until sample analysis.

The plastic compatibility of the Adenovirus formulations was analyzed bythe following methods, as described above: intrinsic proteinfluorescence intensity; hexon protein content by RP-UPLC; and Adenovirusparticle concentration by CE. Protein fluorescence and RP-UPLC wereperformed in triplicate and duplicate, respectively, on all three bagsper formulation. CE was performed for two of the three bags, obtaining asingle reportable value per bag and time point.

Results and Conclusions

The results presented in FIGS. 9-11 clearly show that the Adenovirusformulations 1, 2 and 3 without HBCD are incompatible with plasticduring short-term storage at 25° C. Decreases in intrinsic proteinfluorescence, hexon content, and viral particle concentration areobserved. This indicates that the concentration of (infectious) viralparticles and proteins decreases in the formulations, by about a thirdover the course of one week. As shown in example 1, Ad26 in formulation1 is stable under these conditions, regarding e.g. protein and particlecontent, when stored in a compatible primary packaging material such asglass. Formulation 2 is a stable Adenovirus formulation as described inliterature [2]. The observed incompatibility is thus specific for thebioprocess bag surface.

The presence of HBCD at 5% (w/w) in formulations 1, 2 or 3+HBCD hasprotected the Adenovirus from surface-induced degradation. FIGS. 9-11show that Ad26 in these formulations supplemented with HBCD iscompatible with plastic, regarding protein and particle content. Thesedata show that β-cyclodextrins provide a protective effect againstplastic surface-induced degradation of viruses that are contained inliquid formulations in bioprocess bags. Importantly, since the effect isobserved in all three formulations, despite their differences in pH andexcipients, the effect is specific for HBCD and does not depend on theformulation.

Example 3 Experimental Design and Methodology

In the examples above it was demonstrated that, unexpectedly, virusformulations are incompatible with plastic bioprocessing bags, and thatthe addition of HBCD provides protection against surface-induceddegradation from said bags. In order to determine the concentrationrange in which HBCD protects against surface-induced degradation, thefollowing experiment was performed. An Adenoviral preparation comprisingAd26 adenoviruses (as described in [1]) was concentrated andreformulated by ultrafiltration/diafiltration (using a Sartoslice set-upwith Pellicon XL Biomax 300 filter, Millipore) to formulation 2 asdefined in Table 1. Formulation 2 is a commonly used adenovirusformulation, as described in literature [2]. Subsequently, theformulation was supplemented with 0, 0.2, 1 or 5% (w/w) hydroxypropylβ-cyclodextrin (HBCD). All formulations were brought to the same titerby diluting with formulation buffer 2. Control samples were taken fromall formulations at T=0 before testing the plastic compatibility of thematerial.

In order to evaluate the compatibility with plastic of the Adenovirusformulations with different HBCD concentrations, 4 mL of eachformulation was filled in 5 mL Flexboy® bioprocessing bags (SartoriusStedim Biotech GmbH), in n=2 per formulation. The bags were incubated at25±2° C. for a compatibility study, with time points at 3 and 8 days. Ateach time point, samples were removed from the bags and stored at ≤−65°C. until sample analysis.

The plastic compatibility of the Adenovirus formulations was analyzed bythe following methods, as described above: intrinsic proteinfluorescence intensity; hexon protein content by RP-UPLC; total proteincontent by absorbance at 280 nm; Adenovirus particle concentration byCE; and potency (infectivity) by QPCR-based Potency Assay (QPA).

Results and Conclusions

FIGS. 12-16 show the intrinsic protein fluorescence, the total proteinconcentration by absorbance at 280 nm, the hexon protein content byRP-UPLC, the Adenovirus particle concentration by CE and the potency byQPA, respectively, of the four formulations after 8 days of contact witha plastic bioprocessing bag. At T=0, the protein and particle content ofall formulations was the same, but after incubation there is a cleardrop in these attributes depending on the HBCD concentration in theformulation. The formulation with 5% HBCD is compatible with theplastic, while the formulations with 0 or 0.2% HBCD have degradedconsiderable during contact with the plastic: a 20-30% drop in viralprotein concentration, particle content and potency is observed. Theformulation with 1% HBCD shows some improvement in compatibility, butnot as much as the formulation with 5% HBCD. HBCD can thus protectagainst surface-induced degradation already at a concentration of 1%,but its efficacy increases at higher concentrations.

Above 5% and up to at least 30%, full protection is achieved and theformulations are compatible with plastic. The upper limit of the HBCDconcentration is thus determined by safety and injectability limits.HBCD is highly soluble in aqueous solutions, and the viscosity andthereby the injectability of the formulation remain within workableranges up to an HBCD concentration of 50% [3]. HBCD has a good safetyand tolerability profile, and no side effects were observed afterparenteral administration of up to 24 g of HBCD daily [4]. A solution of30% HBCD has an osmolarity of ca. 200-215 mOsm/L, depending on the molarsubstitution, and can thus be part of an isotonic formulation. Takinginto consideration the factors of compatibility, viscosity andinjectability, safety and tolerability, the range in which HBCD can beapplied to protect against surface-induced degradation is 1% (w/w)-30%(w/w).

REFERENCES

-   1. Zahn, R., et al., Ad35 and ad26 vaccine vectors induce potent and    cross-reactive antibody and T-cell responses to multiple filovirus    species. 2012.-   2. Evans, R. K., et al., Development of stable liquid formulations    for adenovirus-based vaccines. J Pharm Sci, 2004. 93(10): p.    2458-75.-   3. Dusautois, C. and S. Neves, Hydroxypropyl Betacyclodextrin: An    Enabling Technology for Challenging Pharmaceutical Formulations.    Roquette, 2009.-   4. Loftsson, T. and M. E. Brewster, Pharmaceutical applications of    cyclodextrins: basic science and product development. Journal of    pharmacy and pharmacology, 2010. 62(11): p. 1607-1621.

1. A method for protecting a virus from surface-induced degradationwherein said virus is contained in a solution in a bag comprised of aplastic and wherein said method comprises the step of adding aβ-cyclodextrin to said solution at a concentration between about 1%(w/w) to about 30% (w/w).
 2. A method according to claim 1, wherein saidβ-cyclodextrin is selected from the group of dimethyl-β-cyclodextrin,2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin,3-hydroxypropyl-β-cyclodextrin and trimethyl-β-cyclodextrin.
 3. A methodaccording to claim 2, wherein said β-cyclodextrin is2-hydroxypropyl-β-cyclodextrin.
 4. A method according to claim 1,wherein said plastic is selected from the group of Ethylene VinylAcetate, Polyamide and Polyethylene.
 5. A method according to claim 1,wherein said bag is a bioprocess bag.
 6. A method according to claim 1,wherein said virus is an adenovirus. 7-11. (canceled)
 12. A method forprotecting a virus from surface-induced degradation wherein said virusis contained in a solution which is contained in a plastic bioprocessbag, comprising adding a β-cyclodextrin to said solution.
 13. The methodaccording to claim 12, wherein said β-cyclodextrin is selected from thegroup of dimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin andtrimethyl-β-cyclodextrin.
 14. The method according to claim 13, whereinsaid β-cyclodextrin is a 2-hydroxypropyl-β-cyclodextrin.
 15. The methodaccording to claim 12, wherein said plastic is selected from the groupof Ethylene Vinyl Acetate, Polyamide and Polyethylene.
 16. A method forprotecting an adenovirus from surface-induced degradation wherein saidadenovirus is contained in a solution which is contained in an EthyleneVinyl Acetate bioprocess bag, comprising adding a2-hydroxypropyl-β-cyclodextrin to said solution.